The Genomic And Transcriptomic Studies On Isonuclear Alloplasmic Male Sterile Rice (Oryza Sativa L.) Lines

Cytoplasmic male sterility (CMS), is not only valuable for hybrid rice breeding, but also provides insight into the interactions between mitochondria and nuclear genomes in plants. In this study, we investigated the genomic, RNA editing, miRNA and transcriptomic of isonuclear alloplasmic male-sterile lines (TAMSLs) which developed by our lab. We would like to give some insight to the DNA methylation, RNA edting process, and gene expression of IAMSLs, nucleo-cytoplasmic interactions and the roles of miRNA in this process. Therefore, these results can provide some information to the mechanism of CMS and mitochondrial retrograde regulation.1. Cytological observations of IAMSLs found that all CMS lines’anthers stained yellow, shriveled, and had rare inclusion. Iodine-potassium iodide (T2-KI) staining of pollens in the CMS lines showed that they had different wrinkle, manifesting allusion abortion. Carbol fuchine staining showed that the pollens of IAMSLs in meiosis, tetrad and early mononuclear microspore stage are normal, but late mononuclear microspore stage anthers are abortive. These results indicated that the alloplasmic sterile lines may abort from middle mononuclear microspore stage. Identification of SSR and AFLP markers found these IAMSLs have few differences in the level of genomic DNA, and the nuclear replacement rate is generally86%, up to97.3%. DNA methylation analysis showed that the methylation statuses of IAMSLs were similar to Meixiang B after hybriding with Meixiang B, but different CMS line had its specific methylation fragment. Particularly, some new methylation sites were present in alloplasmic sterile lines.2. We examined RNA editing status of the mitochondrial genes cox2, atp6and atp9in five rice IAMSLs to investigate whether different cytoplasmic types affectRNA editing. Although many editing sites were conserved among the three genes, we found that the editingefficiency of certain sites was significantly different between different IAMSLs or between IAMSLs and theircorresponding cytoplasmic donor CMS lines. Furthermore, several editing sites were found to be eitherpresent or absent in certain IAMSLs and their corresponding CMS lines. These results indicate that nuclearloci, as well as unknown editing factors within the mitochondria of different cytoplasmic types, may be involved in RNA editing, and they suggested that RNA editing in plant mitochondria is affected by nucleo-cytoplasmic interactions.Although site C223of the atp9transcript in Meixiang A was edited in root tissues and had no observed significant difference in young panicles, which was the target tissue of the CMS phenotype, possible deleterious effects from such "unedited" ATP9proteins in the CMS line cannot beruled out. Therefore, as one of the mitochondrial ATP synthase subunits, unedited atp9transcripts in the ZD-type CMS line may affect ATP production, and in turn, lead to CMS in rice.3. The microarray analysis of three isonuclear aloplasmic CMS lines and its maintainer line Meixiang B showed that many differential genes were found in IAMSLs compared with Meixiang B. There were1185,1068,1310differential genes in aloplasmic CMS lines XQZ-A/MB, ZS97-A/MB and D62-A/MB, respectively. After annotating of these genes, they were mainly:①genes related of pollen development;②transcript factors related with anther development (such as AP2, MYB, MADS);③Hormne related genes;④ATPase-related genes;⑤mitochondrial and chloroplast genes;⑥protein kinase;⑦heat-shock proteins and transporter and so on. Some of these pollen developmental genes that reported, such as MS2, ABCG15, were also significant down-regulated in the aloplasmic CMS lines. Moreover, the expression patterns of different aloplasmic CMS lines were different, for instant, some differential genes of ZS97-A/MB was different to XQZ-A/MB and D62-A/MB compared with Meixiang B. These results indicated that ZS97-A/MB might have different abortion mechanism.4. According to gene expression analysis between the IAMSLs and its maintainer line Meixiang B using microarray, miRNA targets were predicted by bioinformatics method. Some miRNA predicted to target differentially expressed genes and quantitative PCR were also validation. In this study, interactions between miRNA and target, suggested that some miRNAs are involved in anther development process. Further studies of these regulatory networks will provide a certain foundation in pollen development process.In this study, the analysis of DNA methylation, RNA editing, gene expression and microRNA of rice IAMSLs, provide some information in the process of cytoplasm (or mitochondrial) affect DNA methylation, RNA editing and nuclear gene expression, nuclear-cytoplasmic interaction, as well as miRNA regulation.